Methods and compositions relating to modulation of A20

a technology of a20 and modulation, applied in the field of methods, can solve the problems of cell death, spontaneous inflammation in mice, and inability to know which proteins mediate this cross-talk,

Inactive Publication Date: 2003-09-11
UNIVERSITY OF CHICAGO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Indeed, the inability to inhibit NF-.kappa.B activity results in spontaneous inflammation in mice that lack the constitutive inhibitor of NF-.kappa.B, I.kappa.B.alpha.
However, it is uncertain which proteins mediate this cross-talk between signaling pathways, and whether such cross-talk occurs in innate immune cells.
TNFR engagement can recruit TRADD and FADD proteins to the receptor, ultimately resulting in activation of caspases and death of the cell.
Such methods may result in increasing or decreasing programmed cell death.

Method used

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  • Methods and compositions relating to modulation of A20
  • Methods and compositions relating to modulation of A20
  • Methods and compositions relating to modulation of A20

Examples

Experimental program
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Effect test

example 1

A20 is a Novel Molecule Which Regulates TNF Responses

[0212] To further evaluate the role of macrophage derived cytokines in stimulating intestinal immune responses, the role of the pro-inflammatory cytokine TNF was investigated. TNF is released predominantly from myeloid cells such as macrophages in response to bacterial cell wall polysaccharides such as lipopolysaccharide (LPS), abundant in the microbe-rich intestinal milieu. As described below, dysregulated TNF expression causes intestinal inflammation (Kontoyiannis et al., 1999). It was hypothesized that cellular responses to TNF should also be properly regulated to maintain mucosal immune homeostasis.

[0213] As various lines of evidence suggested that TNF induced signals regulate intestinal inflammation, it was sought to understand the molecular mechanisms by which these signals are regulated. TNF signals, largely mediated through TNFR1, lead to the activation NF-.kappa.B and JNK pathways, as well as caspase mediated PCD pathways...

example 2

Generation and Histology of A20.sup.- / - mice

[0216] To disrupt the A20 gene in mice, the inventors cloned and mapped a 14 kb genomic clone encompassing the murine A20 gene. A gene targeting construct designed to eliminate the ATG start codon and the first 738 base pairs of the coding sequence (corresponding to residues 1-246) was transfected into ES cells (FIG. 2). Correctly targeted ES clones were identified by Southern blotting (FIG. 2) and injected into C57B1 / 6 blastocysts, after which male chimeric mice were bred to C57B1 / 6 females to obtain germline transmission of the A20 mutant allele.

[0217] A20.sup..+-. mice appeared normal without evidence of pathology. A20.sup.- / - mice were born from interbred A20.sup..+-. mice in Mendelian ratios, demonstrating that A20 is not required for embryonic survival. A20.sup.- / - pups were runted as early as one week of age and began to die shortly thereafter (FIG. 3). Gross and histological examination of three to six week old A20.sup.- / - mice rev...

example 3

Sensitivity of A20.sup.- / - Thymocytes and MEFs to TNF Mediated PCD

[0218] The role of A20 in regulating inflammation was further evaluated by examining the sensitivity of A20.sup.- / - mice to LPS. All A20.sup.- / -mice died within two hours of injection of 5 mg / kg LPS, while A20.sup.+ / + and A20.sup..+-. mice given 5, 12, or 25 mg / kg LPS survived without significant morbidity (Table 4). This hypersensitivity to LPS was correlated with increased numbers of A20.sup.- / - splenocytes expressing TNF after LPS stimulation. In addition, A20.sup.- / - mice were highly susceptible to low doses of TNF, as all A20.sup.- / - mice died within two hours of injection of 0.1 mg / kg TNF, while A20.sup.+ / + and A20.sup..+-. mice given 0.1, 0.2, or 0.4 mg / kg TNF survived (Table 4).

4TABLE 4 A20.sup.- / - mice succumb to sub-lethal doses of LPS and TNF*. LPS mg / kg TNF mg / kg 5 12.5 25 0.1 0.2 0.4 + / +, + / - 4 / 4 4 / 4 4 / 4 5 / 5 2 / 2 2 / 2 - / - 0 / 4 -- -- 0 / 4 -- 1 *17-20 day old A20.sup.+ / + and A20.sup.- / - littermates were given t...

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Abstract

The invention provides compositions and methods for treating diseases characterized by aberrant programmed cell death and / or inflammation, comprising mediating A20 function in the subject. Such diseases include Crohn's disease, inflammatory bowel disease, a disease associated with ischemic injury, a toxin-induced liver disease and cancer. The invention further provides methods and compositions for assays for modulators of A20.

Description

[0001] This application claims the priority of U.S. Provisional Patent Application Serial No. 60 / 285,427, filed Apr. 19, 2001, the entire disclosure of which is specifically incorporated herein by reference.[0002] 1. Field of the Invention[0003] The present invention relates generally to the field of medicine. More particularly, it concerns compositions and methods for treating disease conditions comprising modulating A20.[0004] 2. Description of Related Art[0005] Intestinal inflammatory responses to microbial pathogens must be delicately balanced to effectively eliminate pathogens while preventing autoimmunity. The critical roles of cytokines in maintaining this balance have been demonstrated in both experimental models such as gene targeted transgenic mice and in human IBD patients. (Sadlock et al., 1993; Willerford et al., 1995; Kuhn et al., 1993; Blumberg et al., 1999; Fiocchi, 1999). One of the most important cytokines for regulating intestinal inflammation is tumor necrosis fa...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/17G01N33/68
CPCG01N33/6863A61K38/1709
Inventor MA, AVERILBOONE, DAVIDLEE, ERIC
Owner UNIVERSITY OF CHICAGO
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